FIELD OF THE INVENTION
[0001] The present invention relates generally to the fields of genetics and medicine. The
present invention more particularly discloses the identification of a human obesity
susceptibility gene, which can be used for the diagnosis, prevention and treatment
of obesity and related disorders, as well as for the screening of therapeutically
active drugs. The invention more specifically discloses certain alleles of the pancreatic
polypeptide receptor 1
(PPYR1,
NPY4R) gene related to susceptibility to obesity and representing novel targets for therapeutic
intervention. The present invention relates to particular mutations in the PPYR1 gene
and expression products, as well as to diagnostic tools and kits based on these mutations.
The invention can be used in the diagnosis of predisposition to, detection, prevention
and/or treatment of coronary heart disease and metabolic disorders, including hypoalphalipoproteinemia,
familial combined hyperlipidemia, insulin resistant syndrome X or multiple metabolic
disorder, coronary artery disease, diabetes and dyslipidemic hypertension.
BACKGROUND OF THE INVENTION
[0002] Approximately three to eight percent of the total health costs of modem industrialized
countries are currently due to the direct costs of obesity (Wolf, 1996). In Germany,
the total costs (both direct and indirect) related to obesity and comorbid disorders
were estimated at 21 billion German marks in 1995 (Schneider, 1996). By 2030 these
costs will rise by 50% even if the prevalence of obesity does not increase further.
[0003] Obesity is often defined simply as a condition of abnormal or excessive fat accumulation
in adipose tissue, to the extent that health may be impaired. The underlying disease
is the process of undesirable positive energy balance and weight gain. An abdominal
fat distribution is associated with higher health risks than a gynoid fat distribution.
[0004] The body mass index (BMI; kg/m
2) provides the most useful, albeit crude, population-level measure of obesity. It
can be used to estimate the prevalence of obesity within a population and the risks
associated with it. However, BMI does not account for body compositon or body fat
distribution (WHO, 1998).
Table 1: Classification of overweight in adults according to BMI (WHO, 1998)
Classification |
BMI (kg/m2) |
Risk of co-morbidities |
Underweight |
< 18.5 |
Low (but risks of other clinical problems increased) |
Normal range |
18.5 - 24.9 |
Average |
Overweight |
≥ 25 |
|
Pre-obese |
25 - 29.9 |
Increased |
Obese class I |
30 - 34.9 |
Moderate |
Obese class I |
35 - 39.9 |
Severe |
Obese class I |
≥ 40 |
Very severe |
[0005] Obesity has also been defined using the 85
th and 95
th BMI-percentiles as cutoffs for definition of obesity and severe obesity. BMI-percentiles
have been calculated within several populations; centiles for the German population
based on the German National Nutrition Survey have been available since 1994 (Hebebrand
et al., 1994, 1996). Because the WHO classification of the different weight classes
can only be applied to adults, it has become costumary to refer to BMI-percentiles
for the definition of obesity in children and adolescents.
[0006] The recent rise in the prevalence of obesity is an issue of major concern for the
health systems of several countries. In the USA the increase in the prevalence of
all classes of obesity has been dated to the time period approximately between 1976
and 1990. During this time span, the prevalence of obesity increased by more than
one half rising from 14.5% to 22.5%. Similar trends have been observed in other countries
in Europe and South America.
[0007] Children and adolescents have not been exempt from this trend. Quite to the contrary,
the increase in the USA has been substantial. Thus, between the 1960ies and 1990,
overweight and obesity increased dramatically in 6 through to 17 year olds. The increments
translate into relative increases of 40% using the 85
th BMI-centile (calculated in the 1960ies) as a cutoff and 100% upon use of the 95
th centile. In a cross sectional study of German children and adolescents treated as
inpatients for extreme obesity between 1985 and 1995, we have reported a significant
increase of the mean BMI of almost 2 kg/m
2 over this ten year period. Within this extreme group, the increments were most pronounced
in the uppermost BMI ranges.
[0008] The mechanisms underlying this increase in the prevalence of obesity are unknown.
Environmental factors have commonly been invoked as the underlying cause. Basically,
both an increased caloric intake and a reduced level of physical activity have been
discussed. In England the increase in obesity rates has been attributed to the latter
mechanism. Thus, in this country, the average caloric intake even decreased somewhat
within the last two decades, whereas indirect evidence stemming from the increases
in hours spent watching television and from the average number of cars per household
points to reduced levels of physical activity as the relevant causative factor.
[0009] Potentially life-threatening, chronic health problems associated with obesity fall
into four main areas: 1) cardiovascular problems, including hypertension, chronic
heart disease and stroke, 2) conditions associated with insulin resistance, namely
Non-Insulin Dependent Diabetes Mellitus (NIDDM), 3) certain types of cancers, mainly
the hormonally related and large-bowel cancers, and 4) gallbladder disease. Other
problems associated with obesity include respiratory difficulties, chronic musculo-skeletal
problems, skin problems and infertility (WHO, 1998).
[0010] The main currently available strategies for treating these disorders include dietary
restriction, increments in physical activity, pharmacological and surgical approaches.
In adults, long term weight loss is exceptional using conservative interventions.
Present pharmacological interventions typically induce a weight loss of between five
and fifteen kilograms; if the medication is discontinued, renewed weight gain ensues.
Surgical treatments are comparatively successful and are reserved for patients with
extreme obesity and/or with serious medical complications.
[0011] Recently, a 10 year old massively obese girl, in whom a leptin deficiency mutation
had been detected, was treated successfully with recombinant leptin. This is the first
individual who therapeutically profited from the detection of the mutation underlying
her morbid obesity.
[0012] Several twin studies have been performed to estimate heritability of the BMI, some
of which have encompassed over 1000 twin pairs. The results have been very consistent:
The intrapair correlations among monozygotic twins were typically between 0.6 and
0.8, independent of age and gender. In one study, the correlations for monozygotic
and dizygotic twins were basically the same, independent of whether the twins had
been reared apart or together. Heritability of the BMI was estimated at 0.7; non-shared
environmental factors explained the remaining 30% of the variance. Surprisingly, shared
environmental factors did not explain a substantial proportion of the variance. Both
hypercaloric and hypocaloric alimentation lead to similar degrees of weight gain or
loss among both members of monozygotic twin pairs, indicating that genetic factors
regulate the effect of environmentally induced variation of energy availability on
body weight. Metabolic reactions and changes in body fat distribution upon overeating
and undereating are also under genetic control (reviewed in Hebebrand et al., 1998).
[0013] A large adoption study has revealed that the BMI of adoptees is correlated with that
of their biological parents and not with the BMI of the adoptive parents. Depending
on the family study, the correlation between the BMI of sibs is between 0.2 and 0.4.
Parent-offspring correlations are typically slightly lower. Segregation analyses have
repeatedly suggested a major recessive gene effect. Based on these analyses, sample
size calculations have been performed based on both concordant and discordant approaches.
In contrast to the expectations, the concordant sib-pair approach was superior; a
lower number of families were required to achieve the same power.
[0014] Family studies based on extremely obese young index patients, either mother or father
or both, have a BMI > 90
th decile in the vast majority of the families. Based on index patients with a BMI >
95
th centile, approximately 20% of the respective families have a sib with a BMI > 90
th centile.
[0015] In conclusion, it is apparent that environmental factors interact with specific genotypes
rendering an individual more or less susceptible to the development of obesity. Furthermore,
despite the fact that major genes have been detected, it is necessary to consider
that the spectrum reaches from such major genes to genes with an only minor influence.
[0016] The discovery of the leptin gene at the end of 1994 (Zhang et al., 1994) has been
followed by a virtual explosion of scientific efforts to uncover the regulatory systems
underlying appetite and weight regulation. It is currently the fastest growing biomedical
field. This upswing has also resulted in large scaled molecular genetic activities
which, due to obvious clinical interest, are basically all related to obesity in humans,
rodents and other mammals (Hebebrand et al., 1998).
[0017] In this respect, many genes in which mutations lead to the presently known monogenic
forms of obesity have been cloned in rodents. Systemic consequences of these mutations
are currently being analysed. These models have provided insights into the complex
regulatory systems involved in body weight regulation, the best known of which includes
leptin and its receptor.
[0018] In mice, but also in pigs, over 15 quantitative trait loci (QTL) have been identified
that are most likely relevant in weight regulation (Rankinen et al., 2002).
[0019] In humans, four exceedingly rare autosomal recessive forms of obesity have been described
as of 1997. Mutations in the genes encoding for leptin, leptin receptor, prohormone
convertase 1 and pro-opiomelanocortin (POMC) have been shown to cause massive obesity
of an early onset type, associated with hyperphagia. Distinct additional clinical
(e.g. red hair, primary amenorrhea) and/or endocrinological abnormalities (e.g. markedly
altered serum leptin levels, lack of ACTH secretion) pinpointed to the respective
candidate genes. Both the monogenic animal models and the human monogenic forms have
led to new insights into the complex system underlying body weight regulation.
[0020] Very recently, the first autosomal dominant form of obesity was described in humans.
Two different mutations within the melanocortin-4 receptor gene
(MC4R) were observed to lead to extreme obesity in probands heterozygous for these variants.
In contrast to the aforementioned findings, these mutations do not implicate readily
obvious phenotypic abnormalities other than extreme obesity (Vaisse et al., 1998;
Yeo et al., 1998). Interestingly, both groups detected the mutations by systematic
screens in relatively small study groups (n=63 and n=43).
[0021] Hinney et al. (1999) screened the
MC4R in a total of 492 obese children and adolescents. All in all, four individuals with
two different mutations leading to haplo-insufficiency were detected. One was identical
to that previously observed by Yeo et al. (1998). The other mutation, which was detected
in three individuals, induced a stop mutation in the extracellular domain of the receptor.
Approximately one percent of extremely obese individuals harbour haplo-insufficiency
mutations in the
MC4R. In addition to the two forms of haplo-insufficiency, Hinney et al. (1999) also detected
additional mutations leading to both conservative and non-conservative amino acid
exchanges. Interestingly, these mutations were mainly observed in the obese study
group. The functional implications of these mutations are currently unknown.
[0022] The identification of individuals with
MC4R mutations is interesting in the light of possible pharmacological interventions.
Thus, intranasal application of adrenocorticotropin
4-10 (ACTH
4-10), representing a core sequence of all melanocortins, resulted in reduced weight,
body fat mass and plasma leptin concentrations in healthy controls. The question arises
as to how mutation carriers would react to this treatment, which could theoretically
counterbalance their reduced receptor density.
[0023] The involvement of specific genes in weight regulation is further substantiated by
data obtained from transgenic mice. For example, MC4R deficient mice develop early
onset obesity (Huszar et al., 1997).
[0024] Different groups are conducting genome scans related to obesity or dependent phenotypes
(BMI, leptin levels, fat mass, etc.). This approach appears very promising, because
it is both systematic and model free. In addition, it has already been shown to be
exceptionally successful. Thus, positive linkage results have been obtained even by
analysing comparatively small study groups. More important, some findings have already
been replicated. Each of the following regions has been identified by at least two
independent groups: chromosome 1q32, chromosome 2p21, chromosome 10 and chromosome
20q13 (Rankinen et al., 2002).
SUMMARY OF THE INVENTION
[0025] The present invention now discloses the identification of a human obesity susceptibility
gene, which can be used for the diagnosis, prevention and treatment of obesity and
related disorders, as well as for the screening of therapeutically active drugs. The
invention more specifically demonstrates that certain alleles of the PPYR1 gene on
chromosome 10 are related to susceptibility to obesity and represent novel targets
for therapeutic intervention.
[0026] The invention concerns several SNPs and mutations in the PPYR1 gene which are associated
with obesity. Preferably, said SNPs associated with obesity are SNP718 and SNP826.
[0027] A particular aspect of this invention resides in compositions of matter comprising
a PPYR1 gene or a fragment thereof comprising a nucleotide T at position 152 (SNP
67), a nucleotide A at position 203 (SNP 118), a nucleotide A at position 245 (SNP160),
a nucleotide T at position 800 (SNP715), a nucleotide A at position 801 (SNP716),
a nucleotide A at position 824 (SNP739), a nucleotide A at position 911 (SNP826),
a nucleotide C at position 945 (SNP860), and/or a nucleotide A at position 1114 (SNP1029)
of SEQ ID No 1, or a combination thereof and expression products thereof (e.g., mRNAs,
polypeptides). More particularly, the invention concerns an isolated or purified PPYR1
polypeptide comprising a residue Ser at amino acid position 23, a residue Met at amino
acid position 40, a residue Met at amino acid position 54, a residue Trp or Glu at
amino acid position 239, a residue Met at amino acid position 247, a residue Met at
amino acid position 276, a residue Pro at amino acid position 287, and/or a residue
Arg at amino acid position 343 of SEQ ID No 2 or a combination thereof. The invention
also resides in particular products such as primers, probes, oligonucleotides and
substrates or supports to which said products are immobilized, which are designed
to specifically detect or amplify a PPYR1 gene or gene product.
[0028] The invention also resides in methods of treating obesity and/or associated disorders
in a subject through a modulation of PPYR1 expression or activity. Such treatments
use, for instance, PPYR1 polypeptides, PPYR1 DNA sequences (including antisense sequences
directed at the PPYR1 gene locus, RNAi), anti-PPYR1 antibodies or drugs that modulate
PPYR1 expression or activity.
[0029] The invention also relates to methods of treating individuals who carry deleterious
alleles of the PPYR1 gene, including presymptomatic treatment or combined therapy,
such as through gene therapy, protein replacement therapy or through the administration
of PPYR1 protein mimetics and/or inhibitors.
[0030] A further aspect of this invention resides in the screening of drugs for therapy
of obesity or associated disorder, based on the modulation of or binding to an allele
of PPYR1 gene associated with obesity or associated disorder or gene product thereof.
[0031] The invention further relates to the screening of alteration(s) associated with obesity
or associated disorder in the PPYR1 gene locus in patients. Such screenings are useful
for diagnosing the presence, risk or predisposition to obesity and associated disorders,
and/or for assessing the efficacy of a treatment of such disorders.
[0032] A further aspect of this invention includes antibodies specific of PPYR1 polypeptide
comprising a residue Ser at amino acid position 23, a residue Met at amino acid position
40, a residue Met at amino acid position 54, a residue Trp or Glu at amino acid position
239, a residue Cys at amino acid position 240, a residue Met at amino acid position
247, a residue Met at amino acid position 276, a residue Pro at amino acid position
287, and/or a residue Arg at amino acid position 343 of SEQ ID No 2, fragments and
derivatives of such antibodies, hybridomas secreting such antibodies, and diagnostic
kits comprising those antibodies. More preferably, the invention concerns antibodies
specific of PPYR1 polypeptide comprising a residue Cys at position 240 and/or a residue
Met at position 276 of SEQ ID No 2 or a combination thereof, fragments and derivatives
of such antibodies, hybridomas secreting such antibodies, and diagnostic kits comprising
those antibodies.
[0033] The invention can be used in the diagnosis of predisposition to, detection, prevention
and/or treatment of obesity, coronary heart disease and metabolic disorders, including
hypoalphalipoproteinemia, familial combined hyperlipidemia, insulin resistant syndrome
X or multiple metabolic disorder, coronary artery disease, diabetes and dyslipidemic
hypertension. The invention can also be used in the diagnosis of predisposition to,
detection, prevention and/or treatment of eating disorders such as anorexia.
LEGEND TO THE FIGURES
[0034]
Figure 1 : Graphical presentation of the linkage peak on chromosome 10q11. The curves
depict the linkage results for the GenomeHIP procedure, the dots depict results for
microsatellite markers in the region. The dotted lines correspond to the Lander &
Krygliak thresholds for suggestive evidence and evidence for linkage respectively.
Linkage results obtained for 99 BAC clones on human chromosome 10 after the GenomeHIPR procedure. Each point on the x-axis corresponds to a clone. Several clones are indicated
by their library name for better orientation (e.g. RP11-70B16). The two horizontal
lines at 3*10-4 and 2*10-5 for the p-values correspond to the significance levels for significant and suggestive
linkage proposed by Krygliak and Lander for whole genome screens. The two highest
peaks delimit the interval of clones with evidence for linkage after the GenomeHIPR analysis.
Figure 2 : Sequence example showing mutation at nucleotide position 718 of the human
PPY1 recptor coding sequence. Figure 2A : example for a heterozygous mutation after
pre-amplification with specific primer FN1A. The site of the mutation is indicated
by the arrow. The codon triplets are deliminated by the vertical bars. Figure 2B :
example for a heterozygous mutation after amplification with generic primers F5/R5.
As in A the site of the mutation is indicated by the arrow. The codon triplets are
deliminated by the vertical bars. As four alleles are present the mutant A allele
is barely visible under the dominant G peak (small A peak under the G peak with app.
3:1 ratio). Figure 2C : Example for a homozygous mutation after pre-amplification
with specific primer FN1A. Figure 2D : Homozygous wild type sequence for PPY1R after
pre-amplification with specific primer FN1A.
Figure 3 : Mutant receptor binding curves. Mutant receptor binding curves. The curves
show results of competition replacement experiments using human PYY and NPY as competitors
for pancreatic polypeptide (PP). Contructs 1, 2 and 3 correspond to the human PPY1
receptor wild-type, mutant 718 (codon 240, Arg-> Cys) and mutant 826 (codon 276, Val
-> Met) respectively.
Figure 4 : second messanger (camp) response of the wild type and mutant receptors
bearing mutations at positions 718 or 826 respectively. Results of cAMP measurements
comparing the NPY4r wild-type receptor (solid curve) to the two mutant receptors bearing
snp718 (dotted curve, 3rd intracellular loop) and snp826 (broken curve, 6th transmembrane domaine). Y-axis relative decrease of cAMP concentration arbitrary
units.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The present invention discloses the identification of PPYR1 as a human obesity susceptibility
gene. Various nucleic acid samples from 89 families with obesity were submitted to
a particular GenomeHIP process. This process led to the identification of particular
identical-by-descent fragments in said populations that are altered in obese subjects.
By screening of the IBD fragments, we identified the pancreatic polypeptide Y receptor
(PPYR1) gene as a candidate for obesity and related phenotypes. This gene is indeed
present in the critical interval and expresses a functional phenotype consistent with
a genetic regulation of obesity. Several mutated alleles and SNPs of the PPYR1 gene
were also identified, as being correlated to obesity in human subjects. A particular
point mutation in codon 240 (SNP 718 C/T) has been found in up to 60% of the tested
obese subjects and is strongly associated with obesity (table 3). Other mutations
were found, for instance, in codons 23, 40, 54, 99, 239, 247, 276, 287 and 343.
[0036] The present invention thus proposes to use PPYR1 gene and corresponding expression
products for the diagnosis, prevention and treatment of obesity and associated disorders,
as well as for the screening of therapeutically active drugs.
DEFINITIONS
[0037] Obesity and metabolic disorders: Obesity shall be construed as any condition of abnormal
or excessive fat accumulation in adipose tissue, to the extent that health may be
impaired. Associated disorders, diseases or pathologies include, more specifically,
any metabolic disorders, including hypo-alphalipoproteinemia, familial combined hyperlipidemia,
insulin resistant syndrome X or multiple metabolic disorder, coronary artery disease,
diabetes mellitus and dyslipidemic hypertension. The invention may be used in various
subjects, particularly human, including adults, children and at the prenatal stage.
[0038] Within the context of this invention, the PPYR1 gene locus designates all PPYR1 sequences
or products in a cell or organism, including PPYR1 coding sequences, PPYR1 non-coding
sequences (e.g., introns), PPYR1 regulatory sequences controlling transcription and/or
translation (e.g., promoter, enhancer, terminator, etc.), as well as all corresponding
expression products, such as PPYR1 RNAs (e.g., mRNAs) and PPYR1 polypeptides (e.g.,
a pre-protein and a mature protein).
[0039] As used in the present application, the term "PPYR1 gene" designates the human Pancreatic
Polypeptide Receptor 1 gene on human chromosome 10, as well as variants, analogs and
fragments thereof, including alleles thereof (e.g., germline mutations) which are
related to susceptibility to obesity and metabolic disorders. The PPYR1 gene may also
be referred to as the Y4 gene, the NPY4R gene or the PP1 gene.
[0040] The term "gene" shall be construed to include any type of coding nucleic acid, including
genomic DNA (gDNA), complementary DNA (cDNA), synthetic or semi-synthetic DNA, as
well as any form of corresponding RNA. The term gene particularly includes recombinant
nucleic acids encoding PPYR1, i.e., any non naturally occurring nucleic acid molecule
created artificially, e.g., by assembling, cutting, ligating or amplifying sequences.
A PPYR1 gene is typically double-stranded, although other forms may be contemplated,
such as single-stranded. PPYR1 genes may be obtained from various sources and according
to various techniques known in the art, such as by screening DNA libraries or by amplification
from various natural sources. Recombinant nucleic acids may be prepared by conventional
techniques, including chemical synthesis, genetic engineering, enzymatic techniques,
or a combination thereof. Suitable PPYR1 gene sequences may be found on gene banks,
such as Unigene Cluster for PPYR1 (Hs.54426), Unigene Representative Sequence NM_005972,
REFSEQ mRNAs:NM-005972, MIPS assembly: H8600S1, DOTS assembly: DT.406393 and additional
gene/cDNA sequences: U35232, U42387, Z66526.
[0041] A particular example of a PPYR1 gene comprises SEQ ID No: 1.
[0042] The term "PPYR1 gene" includes any variant, fragment or analog of SEQ ID No: 1 or
of any coding sequence as identified above. Such variants include, for instance, naturally-occurring
variants due to allelic variations between individuals (e.g., polymorphisms), mutated
alleles related to obesity, alternative splicing forms, etc. The term variant also
includes PPYR1 gene sequences from other sources or organisms. Variants are preferably
substantially homologous to SEQ ID No:1, i.e., exhibit a nucleotide sequence identity
of at least about 65%, typically at least about 75%, preferably at least about 85%,
more preferably at least about 95% with SEQ ID No:1. Variants and analogs of a PPYR1
gene also include nucleic acid sequences, which hybridize to a sequence as defined
above (or a complementary strand thereof) under stringent hybridization conditions.
Typical stringent hybridisation conditions include temperatures above 30° C, preferably
above 35°C, more preferably in excess of 42°C, and/or salinity of less than about
500 mM, preferably less than 200 mM. Hybridization conditions may be adjusted by the
skilled person by modifying the temperature, salinity and/or the concentration of
other reagents such as SDS, SSC, etc.
[0043] A fragment of a PPYR1 gene designates any portion of at least about 8 consecutive
nucleotides of a sequence as disclosed above, preferably at least about 15, more preferably
at least about 20 nucleotides, further preferably of at least 30 nucleotides. Fragments
include all possible nucleotide length between 8 and 100 nucleotides, preferably between
15 and 100, more preferably between 20 and 100.
[0044] A PPYR1 polypeptide designates any protein or polypeptide encoded by a PPYR1 gene
as disclosed above. The term "polypeptide" refers to any molecule comprising a stretch
of amino acids. This term includes molecules of various length, such as peptides and
proteins. The polypeptide may be modified, such as by glycosylations and/or acetylations
and/or chemical reaction or coupling, and may contain one or several non-natural or
synthetic amino acids. A specific example of a PPYR1 polypeptide comprises all or
part of SEQ ID No:2 or a variant thereof.
DIAGNOSIS
[0045] The invention now provides diagnosis methods based on a monitoring of the PPYR1 gene
locus in a subject. Within the context of the present invention, the term 'diagnosis"
includes the detection, monitoring, dosing, comparison, etc., at various stages, including
early, pre-symptomatic stages, and late stages, in adults, children and pre-birth.
Diagnosis typically includes the prognosis, the assessment of a predisposition or
risk of development, the characterization of a subject to define most appropriate
treatment (pharmaco-genetics), etc.
[0046] A particular object of this invention resides in a method of detecting the presence
of or predisposition to obesity or an associated disorder in a subject, the method
comprising detecting in a sample from the subject the presence of an alteration in
the PPYR1 gene locus. Preferably, said alteration is selected from the group consisting
of a SNP disclosed in Table 2. More preferably, said alteration is selected from the
group consisting of SNP 67, SNP 118, SNP 160, SNP 295, SNP 715, SNP 716, SNP 718,
SNP 739, SNP 826, SNP 860, SNP 1029 or a combination thereof. Still more preferably,
said alteration is selected from the group consisting of SNP 718 and SNP 826 or a
combination thereof. Optionally, said method comprises a previous step of providing
a sample from a subject. Preferably, the presence of an alteration in the PPYR1 gene
locus in said sample is detected through the genotyping of an haplotype. More preferably,
the haplotype comprises a combination of SNP 588, SNP 718 and SNP 826. Alternatively,
the SNP 588 can be replaced in this haplotype by SNP 195, SNP 897, SNP 948 or SNP
1047.
[0047] An other particular object of this invention resides in a method of assessing the
response of a subject to a treatment of obesity or an associated disorder, the method
comprising detecting in a sample from the subject the presence of an alteration in
the PPYR1 gene locus in said sample. Preferably, said alteration is selected from
the group consisting of a SNP disclosed in Table 2. More preferably, said alteration
is selected from the group consisting of SNP 67, SNP 118, SNP 160, SNP 295, SNP 715,
SNP 716, SNP 718, SNP 739, SNP 826, SNP 860, SNP 1029 or a combination thereof. Still
more preferably, said alteration is selected from the group consisting of SNP 718
and SNP 826 or a combination thereof. Optionally, said method comprises a previous
step of providing a sample from a subject. Preferably, the presence of an alteration
in the PPYR1 gene locus in said sample is detected through the genotyping of an haplotype.
More preferably the haplotype comprises a combination of SNP 588, SNP 718 and SNP
826. Alternatively, the SNP 588 can be replaced in this haplotype by SNP 195, SNP
897, SNP 948 or SNP 1047.
[0048] The inventor identified a second copy of the PPYR1 gene near the known PPYR1 gene.
In the present invention, "PPYR1a" will refer to the known PPYR1 gene whereas "PPYR1bis"
("NPY4bis") will refer to the second copy of the PPYR1 gene identified by the inventors.
The methods of detecting the presence of or predisposition to obesity or an associated
disorder or of assessing the response to a treatment of obesity or an associated disorder
in a subject can require a specific amplification of a particular copy of PPYR1 gene,
more particularly PPYR1a. An object of the present invention is the forward primer
FN1A (SEQ ID No 9) which allows a specific amplification of PPYR1a gene (see Examples).
[0049] The alteration may be determined at the level of the PPYR1 gDNA, RNA or polypeptide.
Optionally, the detection is performed by sequencing all or part of the PPYR1 gene
or by selective hybridisation or amplification of all or part of the PPYR1 gene. More
preferably a PPY1R gene specific amplification is carried out before the alteration
identification step.
[0050] An alteration in the PPYR1 gene locus may be any form of mutation(s), deletion(s),
rearrangement(s) and/or insertions in the coding and/or non-coding region of the locus,
alone or in various combination(s). Mutations more specifically include point mutations.
Deletions may encompass any region of two or more residues in a coding or non-coding
portion of the gene locus, such as from two residues up to the entire gene or locus
Typical deletions affect smaller regions, such as domains (introns) or repeated sequences
or fragments of less than about 50 consecutive base pairs, although larger deletions
may occur as well. Insertions may encompass the addition of one or several residues
in a coding or non-coding portion of the gene locus. Insertions may typically comprise
an addition of between 1 and 50 base pairs in the gene locus. Rearrangement includes
inversion of sequences. The PPYR1 gene locus alteration may result in the creation
of stop codons, frameshift mutations, amino acid substitutions, particular RNA splicing
or processing, product instability, truncated polypeptide production, etc. The alteration
may result in the production of a PPYR1 polypeptide with altered function, stability,
targeting or structure. The alteration may also cause a reduction in protein expression
or, alternatively, an increase in said production.
[0051] In this regard, the present invention now discloses several SNPs and mutations in
the PPYR1 gene, which are associated with obesity. These point mutations are reported
in the following table 2.
Table 2
|
Numbering in SEQ ID No:1 |
Polymorphism |
Codon and/or Amino acid change |
Freq % |
SNP 67 |
152 |
C/T |
Pro 23 -> Ser |
0.30 |
SNP 118 |
203 |
G/A |
Val 40 -> Met |
0.30 |
SNP 160 |
245 |
G/A |
Val 54 -> Met |
0.30 |
SNP 195 |
280 |
G/A |
65 |
|
SNP 295 |
380 |
G/T |
Ala99 -> Ser |
|
SNP 588 |
673 |
G/A |
196 |
|
SNP 691 |
776 |
C/T |
231 |
|
SNP 715 |
800 |
C/T |
Arg239 -> Trp |
1 |
SNP 716 |
801 |
G/A |
Arg239 -> Glu |
1.5 |
SNP 718 |
803 |
C/T |
Arg240 -> Cys |
36 |
SNP 739 |
824 |
G/A |
Val 247 -> Met |
0.30 |
SNP 826 |
911 |
G/A |
Va1276 -> Met |
15 |
SNP 860 |
945 |
T/C |
Leu 287 -> Pro |
0.30 |
SNP 897 |
982 |
C/T |
299 |
|
SNP 948 |
1033 |
C/T |
316 |
|
SNP 1029 |
1114 |
C/A |
Ser 343 -> Arg |
0.30 |
SNP 1047 |
1132 |
G/A |
349 |
|
[0052] These point mutations affect the coding region of the PPYR1 gene and result, for
some of them, in an altered amino acid residue in the encoded polypeptide. These point
mutations have been detected in subjects having obesity. Haplotypes were constructed
for all these SNPs to determine the naturally occurring phase for each possible SNP
combination. The results show that SNP 588 is in almost complete linkage disequilibrium
with all other occurring non-coding SNPs and can be used as a reliable denominator
for the different haplotypes involving the amino acid changes. Haplotypes comprising
SNP 588 and any combination of amino acid changing SNPs were then used to test for
association between all the resulting alleles and obesity. The results show that haplotypes
characterized by a point mutation in codon 240 (see table 2 above) are strongly associated
with obesity (tables 3 and 4).
[0053] In a first variant, the method of the present invention comprises detecting the presence
of an altered PPYR1 gene sequence. This can be performed by sequencing all or part
of the PPYR1 gene, polypeptide or RNA, by selective hybridisation or by selective
amplification, for instance.
[0054] A more specific embodiment comprises detecting the presence of a SNP as disclosed
in Table 2 in the PPYR1 gene sequence of a subject, more particularly the detection
of at least one mutation in codon 239, 240 and/or 276 (see table 2 above) or any combination
thereof. More preferably, the method comprises detecting the presence of a mutation
in codon 240 and/or 276. In a specific embodiment, the method comprises detecting
SNPs 715, 716, 718, and/or 826 or any combination thereof in the PPYR1 gene of a subject.
More preferably, the method comprises detecting SNPs 718 and/or 826 in the PPYR1 gene.
[0055] In another variant, the method comprises detecting the presence of an altered PPYR1
RNA expression. Altered RNA expression includes the presence of an altered RNA sequence,
the presence of an altered RNA splicing or processing, the presence of an altered
quantity of RNA, etc. These may be detected by various techniques known in the art,
including by sequencing all or part of the PPYR1 RNA or by selective hybridisation
or selective amplification of all or part of said RNA, for instance. A more specific
embodiment comprises detecting the presence of a mutation as disclosed in Table 2
in the PPYR1 RNA sequence of a subject, particularly the detection of at least one
mutation in codon 239, 240 and/or 276 (see table 2 above) or any combination thereof.
More preferably, the method comprises detecting the presence of a mutation in codon
240 and/or 276. In a specific embodiment, the method comprises detecting SNPs 715,
716, 718, and/or 826 or any combination thereof in the PPYR1 RNA of a subject. More
preferably, the method comprises detecting SNPs 718 and/or 826 in the PPYR1 gene.
[0056] In a further variant, the method comprises detecting the presence of an altered PPYR1
polypeptide expression. Altered PPYR1 polypeptide expression includes the presence
of an altered polypeptide sequence, the presence of an altered quantity of PPYR1 polypeptide,
the presence of an altered tissue distribution, etc. These may be detected by various
techniques known in the art, including by sequencing and/or binding to specific ligands
(such as antibodies), for instance. A more specific embodiment comprises detecting
the presence of a mutation as disclosed in Table 2 in the PPYR1 polypeptide sequence
of a subject, particularly SNPs 715, 716, 718, and/or 826 or any combination thereof.
More preferably, the method comprises detecting SNPs 718 and/or 826 in the PPYR1 gene.
Another embodiment thus comprises the detection of the presence of at least one mutation
in codon 239, 240 and/or 276 (see table 2 above) or any combination thereof in a subject.
More preferably, the method comprises detecting the presence of a mutation in codon
240 and/or 276.
[0057] As indicated above, various techniques known in the art may be used to detect or
quantify altered PPYR1 gene or RNA expression or sequence, including sequencing, hybridisation,
amplification and/or binding to specific ligands (such as antibodies). Other suitable
methods include allele-specific oligonucleotide (ASO), allele-specific amplification,
Southern blot (for DNAs), Northern blot (for RNAs), single-stranded conformation analysis
(SSCA), PFGE, fluorescent in situ hybridization (FISH), gel migration, clamped denaturing
gel electrophoresis, heteroduplex analysis, RNase protection, chemical mismatch cleavage,
ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA).
[0058] Some of these approaches (e.g., SSCA and CGGE) are based on a change in electrophoretic
mobility of the nucleic acids, as a result of the presence of an altered sequence.
According to these techniques, the altered sequence is visualized by a shift in mobility
on gels. The fragments may then be sequenced to confirm the alteration.
[0059] Some others are based on specific hybridisation between nucleic acids from the subject
and a probe specific for wild-type or altered PPYR1 gene or RNA. The probe may be
in suspension or immobilized on a substrate. The probe is typically labelled to facilitate
detection of hybrids.
[0060] Some of these approaches are particularly suited for assessing a polypeptide sequence
or expression level, such as Northern blot, ELISA and RIA. These latter require the
use of a ligand specific for the polypeptide, more preferably of a specific antibody.
[0061] In a particular, preferred, embodiment, the method comprises detecting the presence
of an altered PPYR1 gene expression profile in a sample from the subject. As indicated
above, this can be accomplished more preferably by sequencing, selective hybridisation
and/or selective amplification of nucleic acids present in said sample.
Sequencing:
[0062] Sequencing can be carried out using techniques well known in the art, using automatic
sequencers. The sequencing may be performed on the complete PPYR1 gene or, more preferably,
on specific domains thereof, typically those known or suspected to carry deleterious
mutations or other alterations.
Amplification
[0063] Amplification is based on the formation of specific hybrids between complementary
nucleic acid sequences that serve to initiate nucleic acid reproduction.
[0064] Amplification may be performed according to various techniques known in the art,
such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement
amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques
can be performed using commercially available reagents and protocols. Preferred techniques
use allele-specific PCR or PCR-SSCP. Amplification usually requires the use of specific
nucleic acid primers, to initiate the reaction.
[0065] Nucleic acid primers useful for amplifying sequences from the PPYR1 gene or locus
are able to specifically hybridize with a portion of the PPYR1 gene locus that flank
a target region of said locus, said target region being altered in certain subjects
having obesity or associated disorders and said target regions being provided in Table
2 above. More particularly, the amplification is specific of the PPYR1a gene.
[0066] Specific examples of primers that can be used to amplify the PPYR1 target region
comprising SNP718 have a sequence identical or complementary to a portion of SEQ ID
No:1 located between nucleotide residues 700 to 800 or 805 to 900.
[0067] Primers that can be used to amplify PPYR1 target region comprising other SNPs as
identified in table 2 may be designed based on the sequence of SEQ ID No: 1.
[0068] The invention also relates to a nucleic acid primer, said primer being complementary
to and hybridizing specifically to a portion of a PPYR1 coding sequence (e.g., gene
or RNA) altered in certain subjects having obesity or associated disorders. In this
regard, particular primers of this invention are specific for altered sequences in
a PPYR1 gene or RNA. By using such primers, the detection of an amplification product
indicates the presence of an alteration in the PPYR1 gene locus. In contrast, the
absence of amplification product indicates that the specific alteration is not present
in the sample.
[0069] Primers that can be used to specifically amplify the PPYR1 target region comprising
SNP718 should, for instance, comprise a sequence identical or complementary to a portion
of SEQ ID No:1 located between nucleotide residues 800 and 805. A specific example
of such a primer comprises the sequence CGGTGC (nucleotide residues 800-805 of SEQ
ID No:1 with SNP718).
[0070] The invention concerns a nucleic acid primer, wherein said primer is complementary
to and hybridizes specifically to a portion of a PPYR1 gene or RNA, wherein said portion
comprises, a nucleotide T at position 801 and/or a nucleotide A at position 911 of
SEQ ID No 1.
[0071] Typical primers of this invention are single-stranded nucleic acid molecules of about
5 to 60 nucleotides in length, more preferably of about 8 to about 25 nucleotides
in length. The sequence can be derived directly from the sequence of the PPYR gene
locus. Perfect complementarity is preferred, to ensure high specificity. However,
certain mismatch may be tolerated.
[0072] The invention also concerns the use of a nucleic acid primer or a pair of nucleic
acid primers as described above in a method of detecting the presence of or predisposition
to obesity or an associated disorder in a subject or in a method of assessing the
response of a subject to a treatment of obesity or an associated disorder.
Selective hybridization
[0073] Hybridization detection methods are based on the formation of specific hybrids between
complementary nucleic acid sequences that serve to detect nucleic acid sequence alteration(s).
[0074] A particular detection technique involves the use of a nucleic acid probe specific
for wild-type or altered PPYR1 gene or RNA, followed by the detection of the presence
of a hybrid. The probe may be in suspension or immobilized on a substrate or support
(as in nucleic acid array or chips technologies). The probe is typically labelled
to facilitate detection of hybrids.
[0075] In this regard, a particular embodiment of this invention comprises contacting the
sample from the subject with a nucleic acid probe specific for an altered PPYR1 gene
locus, and assessing the formation of an hybrid. In a particular, preferred embodiment,
the method comprises contacting simultaneously the sample with a set of probes that
are specific, respectively, for wild type PPYR1 gene locus and for various altered
forms thereof. In this embodiment, it is possible to detect directly the presence
of various forms of alterations in the PPYR1 gene locus in the sample. Also, various
samples from various subjects may be treated in parallel.
[0076] Within the context of this invention, a probe refers to a polynucleotide sequence
which is complementary to and capable of specific hybridisation with a (target portion
of a) PPYR1 gene or RNA, and which is suitable for detecting polynucleotide polymorphisms
associated with PPYR1 alleles which predispose to or are associated with obesity or
metabolic disorders. Probes are preferably perfectly complementary to the PPYR1 gene,
RNA, or target portion thereof. Probes typically comprise single-stranded nucleic
acids of between 8 to 1000 nucleotides in length, for instance of between 10 and 800,
more preferably of between 15 and 700, typically of between 20 and 500. It should
be understood that longer probes may be used as well. A preferred probe of this invention
is a single stranded nucleic acid molecule of between 8 to 500 nucleotides in length,
which can specifically hybridise to a region of a PPYR1 gene or RNA that carries an
alteration.
[0077] A specific embodiment of this invention is a nucleic acid probe specific for an altered
(e.g., a mutated) PPYR1 gene or RNA, i.e., a nucleic acid probe that specifically
hybridises to said altered PPYR1 gene or RNA and essentially does not hybridise to
a PPYR1 gene or RNA lacking said alteration. Specificity indicates that hybridisation
to the target sequence generates a specific signal which can be distinguished from
the signal generated through non-specific hybridisation. Perfectly complementary sequences
are preferred to design probes according to this invention. It should be understood,
however, that certain mismatch may be tolerated, as long as the specific signal may
be distinguished from non-specific hybridisation.
[0078] Particular examples of such probes are nucleic acid sequences complementary to a
target portion of the PPYR1 gene or RNA carrying a point mutation as listed in Table
2 above, more preferably a nucleotide T at position 152 (SNP 67), a nucleotide A at
position 203 (SNP118), a nucleotide A at position 245 (SNP160), a nucleotide T at
position 800 (SNP715), a nucleotide A at position 801 (SNP716), a nucleotide A at
position 824 (SNP739), a nucleotide A at position 911 (SNP826), a nucleotide C at
position 945 (SNP860), and/or a nucleotide A at position 1114 (SNP1029) of SEQ ID
No 1 or a combination thereof, still more preferably a nucleotide T at position 800,
a nucleotide A at position 801 and/or a nucleotide A at position 911 of SEQ ID No
1 or a combination thereof.
[0079] The sequence of the probes can be derived from the sequences of the PPYR1 gene and
RNA as provided in the present application. Nucleotide substitutions may be performed,
as well as chemical modifications of the probe. Such chemical modifications may be
accomplished to increase the stability of hybrids (e.g., intercalating groups) or
to label the probe. Typical examples of labels include, without limitation, radioactivity,
fluorescence, luminescence, enzymatic labelling, etc.
[0080] The invention also concerns the use of a nucleic acid probe as described above in
a method of detecting the presence of or predisposition to obesity or an associated
disorder in a subject or in a method of assessing the response of a subject to a treatment
of obesity or an associated disorder.
Specific Ligand Binding
[0081] As indicated above, alteration in the PPYR1 gene locus may also be detected by screening
for alteration(s) in PPYR1 polypeptide sequence or expression levels. In this regard,
a specific embodiment of this invention comprises contacting the sample with a ligand
specific for a PPYR1 polypeptide and determining the formation of a complex.
[0082] Different types of ligands may be used, such as specific antibodies. In a specific
embodiment, the sample is contacted with an antibody specific for a PPYR1 polypeptide
and the formation of an immune complex is determined. Various methods for detecting
an immune complex can be used, such as ELISA, radio-immunoassays (RIA) and immuno-enzymatic
assays (IEMA).
[0083] Within the context of this invention, an antibody designates a polyclonal antibody,
a monoclonal antibody, as well as fragments or derivatives thereof having substantially
the same antigen specificity. Fragments include Fab, Fab'2, CDR regions, etc. Derivatives
include single-chain antibodies, humanized antibodies, poly-functional antibodies,
etc.
[0084] An antibody specific for a PPYR1 polypeptide designates an antibody that selectively
binds a PPYR1 polypeptide, i.e., an antibody raised against a PPYR1 polypeptide or
an epitope-containing fragment thereof. Although non-specific binding towards other
antigens may occur, binding to the target PPYR1 polypeptide occurs with a higher affinity
and can be reliably discriminated from non-specific binding. Preferred antibodies
are specific for particular forms of PPYR1 polypeptide comprising a mutation in codon
23, 40, 54, 239, 240, 247, 276, 287 and/or 343. More preferably, said antibodies are
specific for particular forms of PPYR1 polypeptide comprising a mutation in codon
239, 240 and/or 276.
[0085] In a specific embodiment, the method comprises contacting a sample from the subject
with (a support coated with) an antibody specific for an altered form of a PPYR1 polypeptide,
and determining the presence of an immune complex. In a particular embodiment, the
sample may be contacted simultaneously, or in parallel, or sequentially, with various
(supports coated with) antibodies specific for different forms of a PPYR1 polypeptide,
such as a wild-type and various altered forms thereof.
[0086] Particular examples of such specific ligands are antibodies specific for altered
PPYR1 polypeptide sequence resulting from mutations listed in Table 2 above. More
particularly these ligands are specific of any mutations in codons 23, 40, 54, 239,
240, 247, 276, 287 and 343as listed in table 2 or any combination of those mutations.
More preferably, said ligands are specific of any mutations in codons 239, 240 and/or
276 listed in Table 2 above.
[0087] The diagnosis methods can be performed in vitro, ex vivo or in vivo, preferably in
vitro or ex vivo. They use a sample from the subject, to assess the status of the
PPYR1 gene locus. The sample may be any biological sample derived from a subject,
which contains nucleic acids or polypeptides. Examples of such samples include fluids,
tissues, cell samples, organs, biopsies, etc. Most preferred samples are blood, plasma,
saliva, urine, seminal fluid, etc. Prenatal diagnosis may also be performed by testing
foetal cells or placental cells, for instance The sample may be collected according
to conventional techniques and used directly for diagnosis or stored. The sample may
be treated prior to performing the method, in order to render or improve availability
of nucleic acids or polypeptides for testing. Treatments include, for instant, lysis
(e.g., mechanical, physical, chemical, etc.), centrifugation, etc. Also, the nucleic
acids and/or polypeptides may be pre-purified or enriched by conventional techniques,
and/or reduced in complexity. Nucleic acids and polypeptides may also be treated with
enzymes or other chemical or physical treatments to produce fragments thereof. Considering
the high sensitivity of the claimed methods, very few amounts of sample are sufficient
to perform the assay.
[0088] As indicated, the sample is preferably contacted with reagents such as probes, primers
or ligands in order to assess the presence of an altered PPYR1 gene locus. Contacting
may be performed in any suitable device, such as a plate, tube, well, glass, etc.
In specific embodiments, the contacting is performed on a substrate coated with the
reagent, such as a nucleic acid array or a specific ligand array. The substrate may
be a solid or semi-solid substrate such as any support comprising glass, plastic,
nylon, paper, metal, polymers and the like. The substrate may be of various forms
and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. The contacting
may be made under any condition suitable for a complex to be formed between the reagent
and the nucleic acids or polypeptides of the sample.
[0089] The finding of an altered PPYR1 polypeptide, RNA or DNA in the sample as described
in Table 2 above is indicative of the presence of an altered PPYR1 gene locus in the
subject, which can be correlated to the presence, predisposition or stage of progression
of obesity or metabolic disorders. For example, an individual having a germline PPYR1
mutation has an increased risk of developing obesity or metabolic disorders. The determination
of the presence of an altered PPYR1 gene locus in a subject also allows the design
of appropriate therapeutic intervention, which is more effective and customized. Also,
this determination at the pre-symptomatic level allows a preventive regimen to be
applied.
GENE, VECTORS, RECOMBINANT CELLS AND POLYPEPTIDES
[0090] A further aspect of this invention resides in novel products for use in diagnosis,
therapy or screening. These products comprise nucleic acid molecules encoding a PPYR1
polypeptide, vectors comprising the same, recombinant host cells and expressed polypeptides.
[0091] In this respect, a further object of this invention is an altered PPYR1 gene or a
fragment thereof comprising a nucleotide T at position 152 (SNP 67), a nucleotide
A at position 203 (SNP118), a nucleotide A at position 245 (SNP160), a nucleotide
T at position 800 (SNP715), a nucleotide A at position 801 (SNP716), a nucleotide
A at position 824 (SNP739), a nucleotide A at position 911 (SNP826), a nucleotide
C at position 945 (SNP860), and/or a nucleotide A at position 1114 (SNP1029) of SEQ
ID No 1 or a combination thereof. More preferably, the invention concerns an altered
PPYR1 gene or a fragment thereof comprising a nucleotide T at position 800 (SNP715
in ORF), a nucleotide A at position 801 (SNP716), a nucleotide A at position 824 (SNP739)
of SEQ ID No 1 or a combination thereof. An other object of this invention is a mutated
PPYR1 polypeptide comprising a residue Ser at amino acid position 23, a residue Met
at amino acid position 40, a residue Met at amino acid position 54, a residue Trp
or Glu at amino acid position 239, a residue Met at amino acid position 247, a residue
Met at amino acid position 276, a residue Pro at amino acid position 287, or a residue
Arg at amino acid position 343 of SEQ ID No 2 or a combination thereof. More preferably,
said mutated PPYR1 polypeptide comprises a residue Trp or Glu at amino acid position
239, and/or a residue Met at amino acid position 276 of SEQ ID No 2.
[0092] A further object of this invention is a vector comprising a nucleic acid encoding
a PPYR1 polypeptide according to the present invention. The vector may be a cloning
vector or, more preferably, an expression vector, i.e., a vector comprising regulatory
sequences causing expression of a PPYR1 polypeptide from said vector in a competent
host cell.
[0093] These vectors can be used to express a PPYR1 polypeptide in vitro, ex vivo or in
vivo, to create transgenic or "Knock Out" non-human animals, to amplify the nucleic
acids, to express antisense RNAs, etc.
[0094] The vectors of this invention typically comprise a PPYR1 coding sequence according
to the present invention operably linked to regulatory sequences, e.g., a promoter,
a polyA, etc. The term "operably linked" indicates that the coding and regulatory
sequences are functionally associated so that the regulatory sequences cause expression
(e.g., transcription) of the coding sequences. The vectors may further comprise one
or several origins of replication and/or selectable markers. The promoter region may
be homologous or heterologous with respect to the coding sequence, and provide for
ubiquitous, constitutive, regulated and/or tissue specific expression, in any appropriate
host cell, including for in vivo use. Examples of promoters include bacterial promoters
(T7, pTAC, Trp promoter, etc.), viral promoters (LTR, TK, CMV-IE, etc.), mammalian
gene promoters (albumin, PGK, etc), and the like.
[0095] The vector may be a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc. Plasmid
vectors may be prepared from commercially available vectors such as pBluescript, pUC,
pBR, etc. Viral vectors may be produced from baculoviruses, retroviruses, adenoviruses,
AAVs, etc., according to recombinant DNA techniques known in the art.
[0096] In this regard, a particular object of this invention resides in a recombinant virus
encoding a PPYR1 polypeptide as defined above. The recombinant virus is preferably
replication-defective, even more preferably selected from E1- and/or E4-defective
adenoviruses, Gag-, pol- and/or env-defective retroviruses and Rep- and/or Cap-defective
AAVs. Such recombinant viruses may be produced by techniques known in the art, such
as by transfecting packaging cells or by transient transfection with helper plasmids
or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP
cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective
recombinant viruses may be found for instance in
WO95/14785,
WO96/22378,
US5,882,877,
US6,013,516,
US4,861,719,
US5,278,056 and
WO94/19478.
[0097] A further object of the present invention resides in a recombinant host cell comprising
a recombinant PPYR1 gene or a vector as defined above. Suitable host cells include,
without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such
as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples
include E.coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g.,
Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established
mammalian cell cultures (e.g., produced from fibroblasts, embryonic cells, epithelial
cells, nervous cells, adipocytes, etc.).
[0098] The present invention also relates to a method for producing a recombinant host cell
expressing a PPYR1 polypeptide according to the present invention, said method comprising
(i) introducing in vitro or ex vivo into a competent host cell a recombinant nucleic
acid or a vector as described above, (ii) culturing in vitro or ex vivo the recombinant
host cells obtained and (iii), optionally, selecting the cells which express the PPYR1
polypeptide at their surface.
[0099] Such recombinant host cells can be used for the production of PPYR1 polypeptides
or of membrane preparations comprising such polypeptides, as well as for screening
of active molecules, as described below. Such cells may also be used as a model system
to study obesity and metabolic disorders. These cells can be maintained in suitable
culture media, such as DMEM, RPMI, HAM, etc., in any appropriate culture device (plate,
flask, dish, tube, pouch, etc.).
DRUG SCREENING
[0100] The present invention also provides novel targets and methods for the screening of
drug candidates or leads. The methods include binding assays and/or functional assays,
and may be performed in vitro, in cell systems, in animals, etc.
[0101] The PPYR1 polypeptides according the present invention, more preferably the PPYR1
comprising a residue Cys at amino acid position 240 and/or a residue Met at amino
acid position 276, are particularly interesting for screening agonist of PPYR1. Indeed,
as shown in the examples, the capacity to bind the ligand is the same than the wild
type PPYR1 but the capacity to induce a secondary messenger is decreased. Therefore,
these altered PPYR1 polypeptide are useful for screening strong agonists.
[0102] A particular object of this invention resides in a method of selecting biologically
active compounds, said method comprising contacting in vitro a test compound with
a PPYR1 gene or polypeptide according to the present invention and determining the
ability of said test compound to bind said PPYR1 gene or polypeptide. Binding to said
gene or polypeptide provides an indication as to the ability of the compound to modulate
the activity of said target, and thus to affect a pathway leading to obesity or metabolic
disorders in a subject. In a preferred embodiment, the method comprises contacting
in vitro a test compound with a PPYR1 polypeptide or a fragment thereof according
to the present invention and determining the ability of said test compound to bind
said PPYR1 polypeptide or fragment. The fragment preferably comprises a ligand-binding
site of the PPYR1 polypeptide.
[0103] A particular object of this invention resides in a method of selecting compounds
active on obesity and associated disorders, said method comprising contacting in vitro
a test compound with a PPYR1 polypeptide according to the present invention or a ligand-binding
site-containing fragment thereof and determining the ability of said test compound
to bind said PPYR1 polypeptide or fragment thereof. Preferred compounds are agonists
of PPYR1, i.e., compounds that can bind PPYR1 and mimic the activity of an endogenous
ligand thereof, such as the pancreatic polypeptide (PP). The polypeptide or fragment
may be used in essentially pure form, in suspension, immobilized on a support, or
expressed in a membrane (intact cell, membrane preparation, liposome, etc.).
[0104] In a further particular embodiment, the method comprises contacting a recombinant
host cell expressing a PPYR1 polypeptide according to the present invention with a
test compound, and determining the ability of said test compound to bind said PPYR1
polypeptide at the surface of said cells and/or to modulate the activity of PPYR1
polypeptide.
[0105] The determination of binding may be performed by various techniques, such as by labelling
of the test compound, by competition with a labelled reference ligand, etc. Modulation
of activity includes, without limitation, stimulation of the PPYR1 polypeptide, intemalisation
of the polypeptide, modulation of cellular cAMP levels, modulation of cytoplasmic
calcium levels, modulation of an interaction between PPYR1 and a G protein, etc. More
preferably, the activity of PPYR1 polypeptide is determined through the second messenger
level. Most preferred compounds are agonists of PPYR1, i.e., compounds that can activate
PPYR1 or mimic the activity of a natural ligand thereof. Modulation also includes
antagonistic activity, i.e., the ability to inhibit or compete with a ligand for binding
or activation of the PPYR1 polypeptide.
[0106] In a particular embodiment, the screening assay uses an altered PPYR1 gene or polypeptide,
particularly a PPYR1 gene or polypeptide having a mutation as listed in Table 2, more
preferably a mutation at position 239, 240 or 276 of SEQ ID No 2, still more preferably
a mutation at position 240 or 276 of SEQ ID No 2, or a combination thereof. Indeed,
because such mutants are present in obese subjects, it is important to assess the
activity of test compounds towards these compounds. In a particular embodiment, the
method comprises contacting a test compound with a recombinant cell comprising a PPYR1
gene having a mutation as listed in Table 2, more particularly, a PPYR1 gene having
a mutation at position 239, 240 or 276 of SEQ ID No 2 or a combination thereof, even
more preferably a PPYR1 gene carrying a mutation at position 240 or 276 of SEQ ID
No 2 or a combination thereof. The effect of the test compound may be compared to
the effect of a natural PPYR1 ligand (e.g., PP). Alternatively, the test compound
may be assayed in the presence of a natural PPYR1 ligand (e.g., PP). The compounds
can be selected on the basis of their capacity to restore a PPYR1 activity in said
cells, as measured by calcium release, cAMP release, etc. In this regard, the SNP718
affects codon 240, which participates in the interaction between PPYR1 and G proteins.
Compounds having the capacity to restore or stimulate a functional interaction between
said altered PPYR1 receptor and a G protein would have great therapeutic potential
in the treatment of obesity in subjects having SNP718.
[0107] A further object of this invention resides in a method of selecting biologically
active compounds, said method comprising contacting in vitro a test compound with
a PPYR1 polypeptide according to the present invention and determining the ability
of said test compound to modulate the activity of said PPYR1 polypeptide.
[0108] A further object of this invention resides in a method of selecting biologically
active compounds, said method comprising contacting in vitro a test compound with
a PPYR1 gene according to the present invention and determining the ability of said
test compound to modulate the expression of said PPYR1 gene.
[0109] In an other embodiment, this invention relates to a method of screening, selecting
or identifying active compounds, particularly compounds active on obesity or metabolic
disorders, the method comprising contacting a test compound with a recombinant host
cell comprising a reporter construct, said reporter construct comprising a reporter
gene under the control of a PPYR1 gene promoter, and selecting the test compounds
that modulate (e.g. stimulate or reduce) expression of the reporter gene.
[0110] The above screening assays may be performed in any suitable device, such as plates,
tubes, dishes, flasks, etc. Typically, the assay is performed in multi-wells plates.
Several test compounds can be assayed in parallel. Furthermore, the test compound
may be of various origin, nature and composition. It may be any organic or inorganic
substance, such as a lipid, peptide, polypeptide, nucleic acid, small molecule, etc.,
in isolated or in mixture with other substances. The compounds may be all or part
of a combinatorial library of products, for instance.
PHARMACEUTICAL COMPOSITIONS, THERAPY
[0111] A further object of this invention is a pharmaceutical composition comprising (i)
a PPYR1 polypeptide, a nucleic acid encoding a PPYR1 polypeptide, a vector or a recombinant
host cell as described above and (ii) a pharmaceutically acceptable carrier or vehicle.
[0112] The invention also relates to a method of treating or preventing obesity or an associated
disorder in a subject, the method comprising administering to said subject a functional
(e.g., wild-type) PPYR1 polypeptide or a nucleic acid encoding the same.
[0113] An other embodiment of this invention resides in a method of treating or preventing
obesity or an associated disorder in a subject, the method comprising administering
to said subject a compound that modulates, preferably that activates or mimics, expression
or activity of a PPYR1 gene or protein according to the present invention.
[0114] A particular embodiment of the present invention resides in a method of treating
or preventing obesity or an associated disorder in a subject, the method comprising
(i) detecting in a sample from the subject the presence of an alteration in the PPYR1
gene locus and (ii) administering to said subject a compound that activates PPYR1.
Preferably, said alteration is selected from the group consisting of a SNP disclosed
in Table 2. More preferably, said alteration is selected from the group consisting
of SNP 67, SNP 118, SNP 160, SNP 295, SNP 715, SNP 716, SNP 718, SNP 739, SNP 826,
SNP 860, SNP 1029 or a combination thereof. Still more preferably, said alteration
is selected from the group consisting of SNP 718 and SNP 826 or a combination thereof.
Optionally, said method comprises a previous step of providing a sample from a subject.
Optionally; said method comprises a step of specifically amplifying PPYR1a gene. Preferably,
said compound is a PPYR1 agonist.
[0115] The invention also concerns the use of a compound that activates PPYR1 for the in
the manufacture of a pharmaceutical composition for treating or preventing obesity
or an associated metabolic disorder in a subject presenting an alteration in the PPYR1
gene locus. Preferably, said alteration is selected from the group consisting of a
SNP disclosed in Table 2. More preferably, said alteration is selected from the group
consisting of SNP 67, SNP 118, SNP 160, SNP 295, SNP 715, SNP 716, SNP 718, SNP 739,
SNP 826, SNP 860, SNP 1029 or a combination thereof. Still more preferably, said alteration
is selected from the group consisting of SNP 718 and SNP 826 or a combination thereof.
Preferably, said compound is a PPYR1 agonist.
[0116] The invention also relates, generally, to the use of a functional PPYR1 polypeptide,
a nucleic acid encoding the same, or a compound that modulates expression or activity
of a PPYR1 gene or protein according to the present invention, in the manufacture
of a pharmaceutical composition for treating or preventing obesity or an associated
metabolic disorder in a subj ect.
[0117] The present invention demonstrates the correlation between obesity (and related disorders)
and an alteration in the PPYR1 gene locus. The invention thus provides a novel target
of therapeutic intervention. Various approaches can be contemplated to restore or
modulate the PPYR1 activity or function in a subject, particularly those carrying
an altered PPYR1 gene locus. Supplying wild-type function to such subjects is expected
to suppress phenotypic expression of obesity and associated disorders in a pathological
cell or organism. The supply of such function can be accomplished through gene or
protein therapy, or by administering compounds that modulate or mimic PPYR1 polypeptide
activity (e.g., agonists as identified in the above screening assays).
[0118] The wild-type PPYR1 gene or a functional part thereof may be introduced into the
cells of the subject in need thereof using a vector as described above. The vector
may be a viral vector or a plasmid. The gene may also be introduced as naked DNA.
The gene may be provided so as to integrate into the genome of the recipient host'
cells, or to remain extra-chromosomal. Integration may occur randomly or at precisely
defined sites, such as through homologous recombination. In particular, a functional
copy of the PPYR1 gene may be inserted in replacement of an altered version in a cell,
through homologous recombination. Further techniques include gene gun, liposome-mediated
transfection, cationic lipid-mediated transfection, etc. Gene therapy may be accomplished
by direct gene injection, or by administering ex vivo prepared genetically modified
cells expressing a functional PPYR1 polypeptide.
[0119] Other molecules with PPYR1 activity (e.g., peptides, drugs, PPYR1 agonists, or organic
compounds) may also be used to restore functional PPYR1 activity in a subject or to
suppress the deleterious phenotype in a cell.
[0120] Restoration of functional PPYR1 gene function in a cell may be used to prevent the
development of obesity or metabolic disorders or to reduce progression of said diseases.
Such a treatment may suppress the obese phenotype of a cell, particularly those cells
carrying a deleterious allele.
[0121] In an alternative, the invention resides in methods of treating eating disorders,
more preferably anorexia, in a subject through a modulation of PPYR1 expression. For
example, in a subject who is heterozygous for PPYR1 with one altered allele, the expression
of the wild type allele can be partially or completely inhibited (i.e. by RNAi or
antisense).
[0122] Further aspects and advantages of the present invention will be disclosed in the
following experimental section, which should be regarded as illustrative and not limiting
the scope of the present application.
EXAMPLES
1. Identification of an Obesity susceptibility locus on human chromosome 10
A. Linkage studies
[0123] Hager et al. (1998) first identified a locus on human chromosome 10 linked to massive
human obesity. The linkage study showed evidence for linkage between markers D10S191
and D10S220 with a MLS maximum at marker D10S197. The whole region covered an interval
of approximately 39 centimorgan.
The locus on chromosome 10p may account for 21-36 % of the obesity in the study group
analysed by the French group (Hager et al., 1998).
Hinney at al. (2000) replicated the linkage to chromosome 10. For this purpose, 94
German families with two obese children were genotyped for 12 microsatellite markers
in the region previously identified by Hager et al. (1998). The MLS is 2.3. A further
study (Price RA, 2001) independently confirmed linkage of this locus in an American
population. It is therefore currently safe to conclude that a substantial number of
obese families harbour (an) allele(s) predisposing to obesity in a gene on chromosome
10.
B. GenomeHIP platform to identify the chromosome 10 susceptibility gene
[0124] As outlined above, the chromosomal interval of the initial linkage findings is huge
(39 cM), not allowing a positional cloning approach to identify the obesity susceptibility
gene in this region. We applied our GenomeHIP platform to reduce the region to a small
interval that would allow rapid identification of the obesity susceptibility gene.
Briefly, the technology consists of forming pairs from the DNA of related individuals.
Each DNA is marked with a specific label allowing its identification. Hybrids are
then formed between the two DNAs. A particular process (
WO00/53802) is then applied that selects all fragments identical-by-descent (IBD) from the two
DNAs in a multi step procedure. The remaining IBD enriched DNA is then scored against
a BAC clone derived DNA microarray that allows the positioning of the IBD fraction
on a chromosome.
The application of this process over many different families results in a matrix of
IBD fractions for each pair from each family. Statistical analyses then calculate
the minimal IBD regions that are shared between all families tested. Significant results
(p-values) are evidence for linkage of the positive region with the trait of interest
(here obesity). The linked interval can be delimited by the two most distant clones
showing significant p-values.
In the present study, 89 families of German origin (117 independent sib-pairs) concordant
for massive obesity (as defined by a body mass index > 90%ile) were submitted to the
GenomeHIP process. The resulting IBD enriched DNA fractions were then labelled with
Cy5 fluorescent dyes and hybridised against a DNA array consisting of 99 chromosome
10 derived human BAC clones resulting in an average chromosome-wide resolution of
250 kilobases. Non-selected DNA labelled with Cy3 was used to normalise the signal
values and compute ratios for each clone. Clustering of the ratio results were then
performed to determine the IBD status for each clone and pair.
By applying this procedure, several BAC clones spanning an approximately 4 mega-base
region on chromosome 10 (bases 44000000 to 48000000) were identified, that showed
significant evidence for linkage to obesity (table 2).
Table 3: Linkage results for chromosome 10q11 in the region of PPY1R: Indicated is
the region correspondent to 4 BAC clones with evidence for linkage. The start and
stop positions of the clones correspond to their genomic location respective to the
start of the chromosome (p-ter).
Chr. |
Clone |
cMorgan |
% genotypes |
p- value |
Start |
Stop |
10 |
RP11-358L16-UA221 |
14.03 |
96.55 |
0.987 |
|
|
10 |
RP11-534N5-UA221 |
14.1 |
93.1 |
0.632 |
|
|
10 |
RP11-175I17-UA221 |
14.1 |
91.38 |
0.114 |
|
|
10 |
RP11-556L1-UA221 |
14.21 |
74.14 |
0.000491 |
46466063 |
46637992 |
10 |
RP11-314P12-UA221 |
14.5 |
89.66 |
0.0014 |
46942672 |
47017702 |
10 |
RP11-508N16-UA221 |
15.73 |
82.76 |
0.0000010* |
48319411 |
48319498 |
10 |
RP11-70E21-UA221 |
16.39 |
67.24 |
0.000013* |
49276409 |
49449647 |
C. Identification of an obesity susceptibility gene on chromosome 10
[0125] By screening the aforementioned 4 mega-base chromosomal region, we identified the
pancreatic polypeptide Y receptor (PPYR1) gene as a candidate for obesity and related
phenotypes. This gene is indeed present in the critical interval, with evidence for
linkage delimited by the clones outlined in B. above.
[0126] PPYR1 is an intronless gene that encodes a predicted 375-amino acid polypeptide having
highest homology to the Y1 receptor gene (42% amino acid identity). PPY1R is a member
of the neuropeptide Y receptor family that so far comprises 5 members identified in
humans (NPY1R, NPY2R, NPY4R (PPY1R), NPY5R and NPY6R) that show specific affinities
to a family of neuropeptides (NPY, PPY and PP). Almost all these peptides and receptors
have been shown to play a role in regulating energy metabolism (food intake and energy
expenditure regulation). Of all the receptors PPY1R is the only one that is primarily
expressed in peripheral tissues (gut, adipose tissue) while the others are primarily
expressed in different parts of the brain. It has been shown in several studies that
impairment of the PPY1 receptor is involved in malfunction of food intake regulation
and weight gain in animal studies. The physiological role of the peripheral PPY1 receptor
seems to lie in an inhibitory signal following binding of its physiological ligand
pancreatic polypeptide (PP) thereby activating a cascade that inhibits food-intake
in response to this satiety factor. Mutations in the receptor could therefore decrease
the impact of the satiety factor PP and be involved in the aetiology of obesity.
[0127] Taken together, the linkage results provided in the present application, identifying
the human PPYR1 gene in the critical interval of genetic alterations linked to obesity
on chromosome 10, with its putative involvement in the NPY/PP signalling pathways,
we conclude that alterations (e.g., mutations and/or polymorphisms) in the PPYR1 gene
or its regulatory sequences may contribute to the development of human obesity and
represent a novel target for diagnosis or therapeutic intervention.
2. SNP identification
[0128] 90 families with at least one individual wit extreme, early onset obesity and their
parents were included in the mutation screen. In addition, 10 multigenerational pedigrees
from the CEPH pool of families were screened for mutations in the PPYR1 gene to allow
the unambiguous determination of multi-SNP haplotypes.
[0129] Primers were designed to amplify the single exon of the PPYR1 gene in three overlapping
fragments. The sequence of the primers is provided below:
primer |
sequence |
annealing |
fragment |
SEQ ID |
Reverse 5 |
TAAGTGGAAGAGCCTGGCA |
60 °C |
|
3 |
Forward 5 |
CCTGGAGAATGTCTTCCACAA |
60 °C |
size:642 pb |
4 |
Reverse 6 |
AGGTGGTGTAGATGGTGCG |
60 °C |
|
5 |
Forward 6 |
TTCTCTGAACATTGCCAGGAT |
60 °C |
size:561 pb |
6 |
Reverse 7 |
AGTCCATGATGGTGTAGACGG |
60 °C |
|
7 |
Forward 7 |
AGCATGCAACAAGTGCTTCTAA |
60 °C |
size:568 pb |
8 |
[0130] The resulting amplification products were directly sequenced by dye-terminator sequencing
to identify mutations and polymorphisms in the gene. Ambiguous samples were re-amplified
and re-sequenced by dye-terminator and dye-primer sequencing.
[0131] A total of 12 single nucleotide polymorphisms were detected in the gene (for positions
see table 2). Four of these resulted in changes of the amino-acids in the respective
codons, as illustrated in table 2.
[0132] Haplotypes for all 12 SNPs were constructed to identify the phase for all SNPs. Several
SNPs (see haplotye listing) were in complete linkage disequilibrium (LD). For these,
one SNP (SNP 588) representative for the LD group was selected for further analyses
of the haplotypes. Finally three SNPs were selected for haplotype association analysis.
Haplotypes were analysed in two ways:
for linkage as a multi-allelic marker and
for association using all haplotypes identified to carry out a transmission disequilibrium
test (TDT).
[0133] The TDT is a powerful association test as it is insensitive to population stratification
problems in the tested sample. Briefly, the segregation of alleles from heterozygous
parents to their affected off-spring is tested. The portion of alleles transmitted
to the affected offspring compared to the non-transmitted alleles is compared to the
ratio expected under random distribution. A significant excess of allele transmission
over the expected value is evidence for an association of the respective allele with
the phenotype (here obesity) in question.
[0134] Briefly 400 trio families were genotyped for three SNPs (588, 718 and 826). Haplotypes
were constructed using the GENEHUNTER package for TDT (version 2.1_r2). The results
of this analysis show that certain alleles or haplotypes, all characterized by the
presence of a mutation at position 718 in the coding region (codon 240 that results
in an amino acid change from Arg to Cys) are strongly associated with obesity. In
the tested population, haplotypes having SNP718 are strongly correlated to obesity
(132 transmitted vs 90 non-transmitted, Chi2 = 7.6, p = 0.004, as determined by TDT),
while haplotypes devoid of SNP718 are not transmitted preferentially to obese subjects
(Chi2 = 2.48, p= 0.12). Indeed, haplotypes that do not carry SNP718 show a tendency
to be under-represented in obese subjects (95 transmitted vs 118 non-transmitted),
strengthening the statistical evidence for this mutation to be involved in the development
of obesity in these subjects. Examples of haplotypes with preferential transmission
of SNP718 to obese individuals are given in table 3.
[0135] The SNPs in codon 239 leading to changes of the amino acid as described in table
2are rarer than SNP718 but also show a statistical tendency to be preferentially transmitted
to obese subjects.
Table 4
Haplotype # |
Haplotype code |
SNP Sequence |
transmitted |
Non-transmitted |
HAPLO_1 |
1 1 1 |
ACG |
95 |
118 |
HAPLO_2 |
1 1 2 |
ACA |
4 |
5 |
HAPLO_3 |
2 1 1 |
G C G |
75 |
82 |
HAPLO_4 |
2 1 2 |
G C A |
57 |
54 |
|
|
|
|
|
HAPLO_5 |
221 |
GTG |
132 |
90 |
HAPLO_6 |
222 |
G T A |
7 |
7 |
|
|
|
|
|
[0136] Legend: haplotypes are listed in 5' - 3' direction. 1 and 2 denote the two nucleotide
possibilities for each SNP as listed in table 2. The positions 1-3 of the SNPs listed
correspond to SNPs 588, 718 and 826 respectively as listed in table 2. Other SNPs
were not included because they were in complete linkage disequilibrium with at least
one of the seven SNPs in the haplotype. SNPs representing rare mutations were not
tested individually as their low frequency does not permit meaningful association
studies a list of these mutations is shown in table 3 with their respective frequencies
in the study group.
PCR conditions for locus specific PCR and nested PCR
[0137] Locus specific PCR was performed for each individual to specifically amplify only
the PPYR1a or PPYR1bis locus. The PPYR1a locus was amplified using the reverse primer
FN1A (5'-CAGGGCTCTAGCACATGAAT-3' SEQ ID No 9) in combination with the forward primer
7 (SEQ ID No 8) and the PPYR1bis locus was amplified using the reverse primer FN1G
(5'-CAGGGCTCTAGCACATGAAC-3' SEQ ID No 10) in combination with the same forward primer
7. Amplification reactions were performed in 96 well plates in a 25 µl reaction volume
on a DYAD (MJ Research) using the Expand Long Template PCR System (Roche Molecular
Biochemicals), 7.5 pmol each forward and reverse primer, 175 µM dNTPs, 1.25 units
Expand enzyme mix (Roche Molecular Biochemicals) and 25 ng genomic DNA. After 3 min
at 94°C, amplification was carried out for 35 cycles comprising of 30 sec at 94°C,
30 sec at 58°C, 4 min at 68°C, followed by incubation at 72°C for 10 min.
[0138] The reactions of the locus specific PCR were diluted 1:100 in water in a 96 well
plate. An aliquot of 2 µl of the diluted locus specific PCR reactions was used in
nested PCR reactions with primers F5 (SEQ ID No 3) and R5 (SEQ ID No 4). PCR reactions
were performed in 96 well plates in a 20 µl volume on a Perkin Elmer 9600 containing
1 x GeneAMP PCR buffer II (Applied Biosystems), 5 pmol each forward and reverse primer,
200 µM dNTPs, 2 mM MgCl
2, 1.25 units AmpliTaq Gold polymerase 'Applied Biosystems) and 2 µl 1:100 diluted
locus specific PCR product. After heating at 94°C for 5 min, amplification was carried
out for 30 cycles. Each cycle comprises: 30 sec at 94°C, 30 sec at 60°C, and 40 sec
at 72°C, followed by a final incubation for 10 min at 72°C.
Functional studies of PPYR1 receptor mutations 718 and 826
1) PCR
[0139] Amplification of NPY4(a/bis) receptor coding sequence from 3 different genomic DNAs
by PCR (each individual being homozygous for either position 718 or 826 of the coding
sequence of NPY4a receptor respectively). The goal was to obtain 3 different constructs:
One with the wild type sequence at positions 718 and 826 (718-C and 826-G)
One with the variation at position 718 (718-T, Arg-Cys)
One with the variation at the position 826 (826-A (Val-Met)
[0140] The forward primer contains (from 5' to 3'):
- a non-specific-tailing-sequence (6 bases)
- the sequence of the restriction site Xho I (CTGGAG)
- and 12 bases complementary to the start coding sequence including the ATG start codon
(tccactATGaac)
[0141] The reverse primer contains (from 5' to 3'):
- a non-specific-tailing-sequence (6 bases)
- the sequence of the restriction site Pme I (GTTTAAAC)
- and 20 bases downstream of the TAA codon, starting 7 bases after this stop codon.
PCR was performed using Expand Taq Polymerase from Roche Diagnostics.
2) Digestion of PCR products
[0142] The obtained PCR products (around 1200 bp) were digested with Xho I and PmeI simultaneously
overnight at 37°C and purified using the Qiaquick PCR purification kit.
3) Preparation of the vector (pcDNA3.1/myc-HIS version A, from Invitrogen N°V800-20).
[0143] This vector was digested with Xho I and PmeI simultaneously overnight at 37°C. The
digested vector was loaded on an agarose gel, and the band corresponding to the digested
vector (5400 bp) was excised from the gel. The vector was then purified by using the
Qiaquick gel extraction kit.
4) Ligation
[0144] The digested PCR fragments and the digested vector were ligated using the T4 DNA
Ligase (from Promega) overnight at 4°C.
5) Transformation
[0145] A competent E Coli strain (JM109) was transformed with the ligation mixtures using
a heat shock method (30 min on ice, heat 30sec at 42°c and 1 min on ice). The selection
of transformed bacteria was carried out on LB plates containing ampicillin.
6) Screening of clones
[0146] The clones were screened by colony-PCR using the primers T7 Forward and BGH Reverse.
Only the clones that contained PCR products of the expected length (1.2 kb) were conserved.
7) Sequencing step
[0147] The conserved clones were sequenced using the same primers. For each construct, one
clone containing the expected sequence was conserved.
8) Plasmid Purification
[0148] One positive clone for each construct was selected and a classical Midiprep (Qiagen
kit) was carried out. The DNA concentration was established by UV spectro-photometry
analysis, and 150µg of each construct was precipitated in 150 µL Ethanol (100%).
Functional Evaluation
[0149] For the three samples (wt plus two mutant alleles) precipitate was collected (supernatant
was saved) and dissolved in 150 ul water. (Expected conc 1ug/ul.)
[0150] One plate of COS cells was transfected for each construct with 12 ul (supposedly
12 ug) plasmid to obtain a transient cell line for each construct. Plasmid (12 ul)
cleavage was started with SspI. Transfected cells were harvested and frozen at -80.
[0151] Test binding was performed with different membrane dilutions to confirm binding and
to determine what dilution would be necessary. Radioligand: iodinated hPP.
[0152] Receptor membrane quantities were determined for competition experiments.
[0153] Transient transfections. Two plates for each plasmid based upon new determination
of concentration. Larger volumes of plasmed solutions. Transfected quantities:
Construct 1: 1.4 ug
Construct 2: 1.4 ug
Construct 3: 1.8 ug
[0154] pUC control plasmid: 0.1ug/ul as expected, thus measurements were judged reliable.
[0155] Concentrations were verified on agarose gel.
Plasmids were transformed into competent bacteria, strain DH5-alpha, to be able to
prepare plasmid DNA for additional transfections.
[0156] Plasmid maxi preps performed. Final volume 400 ul.
Construct 1: 1.4 ug/ul
Construct 2: 1.3 ug/ul
Construct 3: 1.3 ug/ul
[0157] Competition experiments: One competition curve for each construct (duplicate samples).
Radioligand hPP. Competing ligand hPYY.
Competition experiments on membranes from transfection 2: One curve for each construct
(including hY4-pTEJ) and each of the ligands hPP, hPYY and hNPY.
[0158] Evaluation of competition experiments (see table 5 and Fig 3).
Conclusion:
No apparent difference between the two mutants and the wildtype. Construct 2 gave quite variable results
for hPYY with very high SEM. The experiment has been repeated 6 times.
Table 5 : Competition Statistics
|
Ki |
±SEM |
n |
hPP |
|
|
|
Construct 1 |
5.4e-11 |
1.8e-12 |
3 |
Construct 2 |
5.1e-11 |
4.5e-12 |
3 |
Construct 3 |
5.1e-11 |
1.6e-12 |
3 |
hPYY |
|
|
|
Construct 1 |
2.3e-9 |
4.3e-11 |
3 |
Construct 2 |
1.9e-8 |
6.6e-9 |
6 |
Construct 3 |
2.3e-9 |
2.8e-10 |
3 |
hNPY |
|
|
|
Construct 1 |
2.1e-7 |
4.6e-8 |
3 |
Construct 2 |
1.6e-7 |
2.5e-8 |
3 |
Construct 3 |
2.0e-7 |
1.5e-9 |
3 |
Construct 1 wildtype
Construct 2 C718T = R240C (in IL3)
Construct 3 3G826A = V276M (in TM6) |
[0159] Transfections into CHO cells were performed for stable expression. Approximately
500 ng of each linearized plasmid was transfected.
[0160] Selection of transfected CHO cells for stable expression initiated with G418.
Evaluation of cAMP experiments.
[0161] Results showed that wild type receptor (after stimulation with forskolin) responded
to hPP with 30% reduction of cAMP production. Construct 2 and 3 showed no response
to the binding stimulus anymore.
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